Much has been written about the molecular pathogenesis of Shigella with respect to the genes and gene products involved in their ability to invade epithelial cells, and thereby to cause dysentery (Makino et al, Microb. Pathog., 5:267-274 (1988); Sansonetti et al, Infect. Immun., 35:852-860 (1982); Hale et al, Infect. Immun., 40:340-350 (1983); Pal et al,. J. Clin. Microbiol., 27:561-563 (1989); and Venkatesan et al, Proc. Nat'l. Acad. Sci. U.S.A., 85:9317-9321 (1988)). In contrast, surprisingly little is known of the precise mechanisms by which Shigella cause watery diarrhea.
Although the cardinal feature of the pathogenesis of Shigella flexneri 2a infection involves the invasion of epithelial cells, because Shigella flexneri 2a can cause watery diarrhea, it has been hypothesized that Shigella flexneri 2a also produces an enterotoxin (Rout et al, Gastroenterology, 68:270-278 (1975); and Kinsey et al, Infect. Immun., 14:368-371 (1976)). More specifically, the following observations have suggested the existence of enterotoxins in Shigella flexneri 2a:
1. Clinically in humans, Shigella flexneri 2a infections are usually characterized by a period of watery diarrhea that precedes the onset of scanty dysenteric stools of blood and mucus (DuPont et al, J. Infect. Dis., 119:296-299 (1969); and Stoll et al, J. Infect. Dis., 146:177-183 (1982)). In mild cases, only watery diarrhea may occur, leading to a clinical picture undistinguishable from that due to enterotoxingenic E. coli infection (Taylor et al, J. Infect. Dis., 153:1132-1138 (1986); and Taylor et al, J. Clin. Microbiol., 26:1362-1366 (1988)). PA1 2. When Shigella are fed to monkeys, three clinical syndromes are seen (Route et al, Gastroenterology, 68:270-278 (1975)). Some monkeys develop only dysentery; some exhibit only watery diarrhea and some exhibit watery diarrhea and dysentery. In vivo perfusion studies by Rout et al, Gastroenterology, 68:270-278 (1975), showed that net transport of water into the lumen of the colon occurs in all ill animals. In contrast, only in the jejunum of monkeys with overt watery diarrhea (alone or followed by dysentery) does there occur net secretion of water, sodium and chloride ions; such net transport does not occur in the jejunum of monkeys manifesting dysentery without watery diarrhea. Net secretion in the jejunum was not accompanied by abnormal histological findings in this anatomic site of the small intestine. PA1 3. The net secretion of water and electrolytes into the jejunum of monkeys with watery diarrhea requires the passage of Shigella through the jejunum (Kinsey et al, Infect. Immun., 14:368-371 (1976)). This was demonstrated by bypassing the small intestine and inoculating Shigella directly into the cecum of monkeys. Of 16 monkeys who developed clinical illness, 15 manifested dysentery, ". . . only rarely preceded by mild diarrhea". Net secretion of water and sodium into the colon was recorded in ill monkeys that developed dysentery following intracecal inoculation, while no abnormalities of water or electrolyte transport were observed in the jejunum of the ill animals.
Together, these observations suggest that Shigella elaborate an enterotoxin that elicits secretion early in the infection as the organisms pass through the jejunum.
However, except for the cytotoxin/neurotoxin/enterotoxin elaborated by Shigella dysenteriae (O'Brien et al, Microbiol. Rev., 5:206-220 (1987); Keusch et al, Pharmac. Ther., 15:403-438 (1982); and Fontaine et al, Infect. Immun., 56:3099-3109 (1988)), but not by other Shigella species, little convincing proof has been generated to substantiate the contention that Shigella, other than Shigella dysenteriae, in fact produce enterotoxins.
More specifically, previous attempts in the art to detect enterotoxic activity in supernatants of Shigella flexneri 2a have yielded positive findings in only one instance. O'Brien et al, Infect. Immun., 15:796-798 (1977), partially purified a toxin produced by Shigella flexneri 2a strain M4243 that was 10 detectable in cell-free supernatants. This toxin stimulated fluid production in rabbit ileal loops, but was also cytotoxic for HeLa cells in monolayers and was lethal when inoculated intraperitoneally into mice. Further, it was not necessary to grow the bacteria in Fe.sup.++ -depleted medium in order to detect the enterotoxic activity. In addition, the cytotoxicity of the toxin described by O'Brien et al, supra, was neutralized by anti-sera to Shiga (Shigella dysenteriae 1) toxin.
Enterotoxic activity in cell-free supernatants of Shigella flexneri 2a and 3a was reported by Ketyi et al, Acta Microbiol. Acad. Sci. Hung., 25:165-171 (1978); Ketyi et al, Acta Microbiol. Acad. Sci. Hung., 25:219-227 (1978); and Ketyi et al, Acta Microbiol. Acad. Sci. Hung., 25:319-325 (1978). Filtered ultrasonic lysates of two Shigella flexneri 2a and 3a strains were founds to give rapid fluid accumulation in rabbit ileal loops (4 hour assay). However, the loops showed no fluid accumulation when examined at 18-24 hours after inoculation. Only three loops were inoculated for each of the two test strains, and when examined at 4 hours, only 2/3 for one strain and 1/3 for the other strain were positive. In addition, the Shigella were not cultured in Fe.sup.++ -depleted medium.
In the present invention, it was discovered for the first time that enterotoxic activity, which is clearly dissociated from cytotoxic activity, is expressed by Shigella flexneri 2a in the bacteria-free culture supernatant, and could be detected only after growth of the bacteria in Fe.sup.++ -depleted medium.
It has been reported that when grown in Fe.sup.++ -depleted medium, enteroinvasive Escherichia coli (EIEC) elaborate an enterotoxin (MW circa 68-80 kDa) that causes fluid accumulation in isolated rabbit ileal loops and an electrical response in Ussing chambers (Fasano et al, Infect. Immun., 58:3717-3723 (1990)). Based on the similarities known to exist between enteroinvasive E. coli and Shigella (Levine et al, J. Infect. Dis., 155:377-389 (1987)), it was postulated in the present invention that Shigella flexneri 2a would express an enterotoxin when grown in Fe.sup.++ -depleted medium.
In the present invention, it was unexpectedly disclosed that Shigella flexneri 2a produces two distinct enterotoxins, one encoded by the chromosome, and the other encoded by an invasiveness virulent plasmid. The latter enterotoxin was found in the present invention to be essentially the same as the EIEC enterotoxin.